Basic-helix-loop-helix (bHLH) proteins participate in a variety of devlopmental processes. In vertebrates, bHLH families critical for skeletal myogenesis, neurogenesis and hemopoiesis have been identified. By mutational analysis in mice, the requirement for these genes in proper cell-type specification has been demonstrated. In addition to determining cell fate, bHLH proteins also have roles in the related process of growth control. For example, the bHLH-encoding genes LYL1, SCL/TAL2 have been identified at translocation breakpoints in T-cell leukemias. The full range of processes in which bHLH protein participate and the functional inter-relationships between bHLH families awaits the identification of new members. As a step towards this goal, a yeast assay system has been used to isolate a new family of mouse bHLH proteins. Preliminary structural and functional characterization of these proteins suggests that they form a new class of bHLH proteins with unique properties. A detailed study of this new family is required to define its functional role relative to other known bHLH proteins. The specific aims of this proposal are to: (1) determine a biological function for these new proteins by producing targeted mutations in mice; (2) define the specificity of these protein for bHLH recognition and DNA binding, and test the transcriptional consequences of these interactions; and (3) identify target genes regulated by these bHLH proteins. The advent of a protein interaction strategy for discovering new bHLH families promises to dramatically increase the number of bHLH proteins under study. Results obtained from this project will lend insight into the developmental role of this new family, and provide a framework for classifying and studying other new bHLH proteins.